Comparative genomic analysis revealed genetic divergence between Bifidobacterium catenulatum subspecies present in infant versus adult guts.

BACKGROUND: The two subspecies of Bifidobacterium catenulatum, B. catenulatum subsp. kashiwanohense and B. catenulatum subsp. catenulatum, are usually from the infant and adult gut, respectively. However, the genomic analysis of their functional difference and genetic divergence has been rare. Here, 16 B. catenulatum strains, including 2 newly sequenced strains, were analysed through comparative genomics. RESULTS: A phylogenetic tree based on 785 core genes indicated that the two subspecies of B. catenulatum were significantly separated. The comparison of genomic characteristics revealed that the two subspecies had significantly different genomic sizes (p < 0.05) but similar GC contents. The functional comparison revealed the most significant difference in genes of carbohydrate utilisation. Carbohydrate-active enzymes (CAZyme) present two clustering patterns in B. catenulatum. The B. catenulatum subsp. kashiwanohense specially including the glycoside hydrolases 95 (GH95) and carbohydrate-binding modules 51 (CBM51) families involved in the metabolism of human milk oligosaccharides (HMO) common in infants, also, the corresponding fucosylated HMO gene clusters were detected. Meanwhile, B. catenulatum subsp. catenulatum rich in GH3 may metabolise more plant-derived glycan in the adult intestine. CONCLUSIONS: These findings provide genomic evidence of carbohydrate utilisation bias, which may be a key cause of the genetic divergence of two B. catenulatum subspecies.

Effects of salt stress on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1.

In this study, we examined the effects of different salt stress application methods on the Lactiplantibacillus plantarum LIP-1 freeze-drying survival rate. The application of salt stress during the stationary phase significantly improved Lactiplantibacillus plantarum LIP-1 freeze-drying survival rates (P < 0.05). The indirect application of salt stress via phosphate-buffered saline containing 0.4 mol/L NaCl (NB group) led to significantly higher freeze-drying survival rates compared to when salt stress was directly applied (NA group: the concentration of NaCl is 0.4 mol/L) (P < 0.05). Following exposure to salt stress, Lactiplantibacillus plantarum LIP-1 cells exuded excessive Na+ out of the cell and transported extracellular K+ into the cell, resulting in upregulation of the trkA gene, which is related to K+ transport, thereby significantly upregulating the expression of a lysR-type transcription factor, which increased the cell membrane unsaturated fatty acid content, reducing the degree of cell membrane damage and improving the freeze-drying survival rate. When the concentration of NaCl is 0.4 mol/L, compared with direct salt stress application, indirect application led to higher intracellular pH and ATP content, which effectively reduced DNA and cell membrane damage, respectively. Together, these results demonstrate that appropriate indirect salt stress application can improve Lactiplantibacillus plantarum LIP-1 freeze-drying resistance.

The diversity and composition of the human gut lactic acid bacteria and bifidobacterial microbiota vary depending on age.

Aging is associated with gut microbiota alterations, characterized by changes in intestinal microbial diversity and composition. However, no study has yet focused on investigating age-related changes in the low-abundant but potentially beneficial subpopulations of gut lactic acid bacteria (LAB) and Bifidobacterium. Our study found that the subjects' age correlated negatively with the alpha diversity of the gut bifidobacterial microbiota, and such correlation was not observed in the gut LAB subpopulation. Principal coordinate analysis (PCoA) and analysis of distribution of operational taxonomic units (OTUs) revealed that the structure and composition of the gut bifidobacterial subpopulation of the longevous elderly group were rather different from that of the other three age groups. The same analyses were applied to identify age-dependent characteristics of the gut LAB subpopulation, and the results revealed that the gut LAB subpopulation of young adults was significantly different from that of all three elderly groups. Our study identified several potentially beneficial bacteria (e.g., Bifidobacterium breve and Bifidobacterium longum) that were enriched in the longevous elderly group (P < 0.05), and the relative abundance of Bifidobacterium adolescentis decreased significantly with the increase in age (P < 0.05). Although both bifidobacteria and LAB are generally considered as health-promoting taxa, their age-dependent distribution varied from each other, suggesting their different life stage changes and potentially different functional roles. This study provided novel species-level gut bifidobacterial and LAB microbiota profiles of a large cohort of subjects and identified several age-or longevity-associated features and biomarkers. KEY POINTS: • The alpha diversity of the gut bifidobacterial microbiota decreased with age, while LAB did not change. • The structure and composition of the gut bifidobacterial subpopulation of the longevous elderly group were rather different from that of the other three age groups. • Several potentially beneficial bacteria (e.g., Bifidobacterium breve and Bifidobacterium longum) that were enriched in the longevous elderly group.

Antihyperlipidaemic effect of microencapsulated Lactobacillus plantarum LIP-1 on hyperlipidaemic rats.

BACKGROUND: Previous studies have shown that Lactobacillus plantarum LIP-1 (hereafter LIP-1) has an obvious hypolipidemic effect, and microencapsulated probiotics can ensure the strains live through the gastrointestinal tract. Although there has been much research on both preparation and assessment methods for probiotics microcapsules, most assessments were made in vitro and few were validated in vivo. In this study, the protective effect of microencapsulation and the possible hypolipidemic mechanisms of probiotic LIP-1 were evaluated in rats. Treatments included rats fed on a normal diet, a high-fat diet, and a high-fat diet with an intragastric supplement of either non-microencapsulated LIP-1 cells (NME LIP-1) or microencapsulated LIP-1 (ME LIP-1). Lipid metabolism indicators were measured during the experiment and following euthanasia. RESULTS: Microencapsulation increased survival and colonization of LIP-1 in the colon. ME LIP-1 was superior to NME LIP-1 in reducing cholesterol. The mechanisms behind the hypolipidemic effect exerted by LIP-1 are possibly due to promoting the excretion of cholesterol, improving antioxygenic potentials, enhancing recovery from the injury in the liver, cardiovascular intima and intestinal mucosa, promoting the generation of short-chain fatty acids, and improving lipid metabolism. CONCLUSIONS: This study confirms that microencapsulation provides effective protection of LIP-1 in the digestive system and the role of LIP-1 in the prevention and cure of hyperlipidaemia, providing theoretical support for probiotics to enter clinical applications. © 2019 Society of Chemical Industry.

Metabolomic analysis of significant changes in Lactobacillus casei Zhang during culturing to generation 4,000 under conditions of glucose restriction.

Lactic acid bacteria are being consumed more frequently as awareness of their health benefits has increased. The industrial production of lactic acid bacteria requires a comprehensive understanding of their survival stress, especially regarding changes in metabolic substances in a glucose-limited environment. In the present study, a metabolomic approach was applied to investigate Lactobacillus casei Zhang using cultures from a common ancestor that were permitted to evolve under conditions with normal or glucose-restricted media for up to 4,000 generations. Metabolomic analyses of intracellular and extracellular differential metabolites under De Man, Rogosa and Sharpe broth (2% vol/vol glucose; Oxoid Ltd., Basingstoke, UK) and glucose-restricted (0.02% vol/vol glucose in De Man, Rogosa and Sharpe broth) conditions were performed at generations 0, 2,000, and 4,000 and revealed 23 different metabolites. Myristic acid, ergothioneine, Lys-Thr, and palmitamide contents exhibited significant reductions between 0 and 4,000 generations, whereas nicotinate, histidine, palmitic acid, l-lysine, urocanate, thymine, and other substances increased. The dynamics of the pathways involved in AA metabolism, including glycine, serine, and threonine metabolism, histidine metabolism, lysine degradation, and arginine and proline metabolism, were also a focus of the present study. There were also changes in several other metabolic pathways, including vitamin B6, thiamine, nicotinate, and nicotinamide, according to generation time. Additionally, in the present study we screened for key metabolites involved in the glucose-restricted response and provided a theoretical basis for comprehensively revealing the regulatory mechanisms associated with L. casei Zhang glucose restriction at the metabolic level. These findings also provide novel ideas and methods for analyzing the glucose-restricted stress response at the metabolic level.

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing.

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and α-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.

A Metabolomics Approach Uncovers Differences between Traditional and Commercial Dairy Products in Buryatia (Russian Federation).

Commercially available and traditional dairy products differ in terms of their manufacturing processes. In this study, commercially available and traditionally fermented cheese, yogurt, and milk beverages were analyzed and compared. The metabolomic technique of ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF) in the MSE mode was used in combination with statistical methods, including univariate analysis and chemometric analysis, to determine the differences in metabolite profiles between commercially and traditionally fermented dairy products. The experimental results were analyzed statistically and showed that traditional and commercial dairy products were well differentiated in both positive and negative ion modes, with significant differences observed between the samples. After screening for metabolite differences, we detected differences between traditional milk beverages and yogurt and their commercial counterparts in terms of the levels of compounds such as l-lysine, l-methionine, l-citrulline, l-proline, l-serine, l-valine and l-homocysteine, and of short peptides such as Asp-Arg, Gly-Arg, His-Pro, Pro-Asn. The greatest difference between commercially available and traditional cheese was in the short peptide composition, as commercially available and traditional cheese is rich in short peptides.

Changes in chemical composition, structural and functional microbiome during alfalfa (Medicago sativa) ensilage with Lactobacillus plantarum PS-8.

Improving silage production by adding exogenous microorganisms not only maximizes nutrient preservation, but also extends product shelf life. Herein, changes in the quality and quantity of Lactobacillus plantarum PS-8 (PS-8) -inoculated alfalfa (Medicago sativa) during silage fermentation were monitored at d 0, 7, 14, and 28 (inoculum dose of PS-8 was 1 × 105 colony forming units [cfu]/g fresh weight; 50 kg per bag; 10 bags for each time point) by reconstructing metagenomic-assembled genomes (MAG) and Growth Rate InDex (GRiD). Our results showed that the exogenous starter bacterium, PS-8 inoculation, became the most dominating strain by d 7, and possibly played a highly active role throughout the fermentation process. The pH value of the silage decreased greatly, accompanied by the growth of acid-producing microorganisms namely PS-8, which inhibited the growth of harmful microorganisms like molds (4.18 vs. 1.42 log cfu/g) and coliforms (4.95 vs. 0.66 log most probable number [MPN]/g). The content of neutral detergent fiber (NDF) decreased significantly (41.6% vs. 37.6%; dry matter basis). In addition, the abundance and diversity of genes coding microbial carbohydrate-active enzymes (CAZymes) increased significantly and desirably throughout the fermentation, particularly the genes responsible for degrading starch, arabino-xylan, and cellulose. Overall, our results showed that PS-8 was replicating rapidly and consistently during early- and mid-fermentation phases, promoting the growth of beneficial lactic acid bacteria and inhibiting undesirable microbes, ultimately improving the quality of silage.

Effects of different initial pH values on freeze-drying resistance of Lactiplantibacillus plantarum LIP-1 based on transcriptomics and proteomics.

The growth and the resistance to adverse environments of lactic acid bacteria would be affected by adjusting the initial pH of the medium. In order to explore the effect of changing the initial pH of culture medium on the freeze-drying survival rate of the Lactiplantibacillus plantarum LIP-1, the effect of initial pH on cell membrane fatty acid composition and key enzyme activity were mainly determined, and the internal mechanism was studied by transcriptomics and proteomics methods. We found that compared with initial pH 7.4 group, initial pH 6.8 group could improve the freeze-drying survival rate of the L. plantarum LIP-1. It was possibly due to the lactate dehydrogenase (LDH) was upregulated in the initial pH6.8 group, which led to a rapid decrease in culture pH. To reduce the inhibitory effect of long-term acid environment on growth, the strain upregulated the expression of fatty acid synthesis-related genes and proteins, promoted the relative content of cyclopropane and unsaturated fatty acids, improved integrity of the cell membranes. The adjustment of fatty acid composition maintained the integrity of the cell membrane in a freeze-drying environment to improve the freeze-drying survival rate of the initial pH6.8 group. In addition, the long-term acid environment stimulated a cross-stress tolerance mechanism that significantly upregulated the expression of a cold stress protein. The results indicated that the optimal initial pH of the medium could improve the ability of L. plantarum LIP-1 to resist freeze-drying.

Profiling of the viable bacterial and fungal microbiota in fermented feeds using single-molecule real-time sequencing.

Fermented concentrated feed has been widely recognized as an ideal feed in the animal industry. In this study, we used a powerful method, coupling propidium monoazide (PMA) pretreatment with single-molecule real-time (SMRT) sequencing technology to compare the bacterial and fungal composition of feeds before and after fermentation with four added lactic acid bacteria (LAB) inoculants (one Lactobacillus casei strain and three L. plantarum strains). Five feed samples consisting of corn, soybean meal, and wheat bran were fermented with LAB additives for 3 d. Following anaerobic fermentation, the pH rapidly decreased, and the mean numbers of LAB increased from 106 to 109 colony-forming units (cfu)/g fresh matter. SMRT sequencing results showed that the abundance and diversity of bacteria and fungi in the feed were significantly higher before fermentation than after fermentation. Fifteen bacterial species and eight fungal genera were significantly altered following fermentation, and L. plantarum was the dominant species (relative abundance 88.94%) in the post-fermentation group. PMA treatment revealed that the bacteria Bacillus cereus, B. circulans, Alkaliphilus oremlandii, Cronobacter sakazakii, Paenibacillus barcinonensis, and P. amylolyticus (relative abundance >1%) were viable in the raw feed. After fermentation, their relative abundances decreased sharply to <0.2%; however, viable L. plantarum was still the dominant species post fermentation. We inferred that our LAB additives grew rapidly and inhibited harmful microorganisms and further improved feed quality. In addition, coupling PMA treatment with the Pacific Biosciences SMRT sequencing technology was a powerful tool for providing accurate live microbiota profiling data in this study.

Bacterial Microbiota and Metabolic Character of Traditional Sour Cream and Butter in Buryatia, Russia.

Traditional sour cream and butter are widely popular fermented dairy products in Russia for their flavor and nutrition, and contain rich microbial biodiversity, particularly in terms of lactic acid bacteria (LAB). However, few studies have described the microbial communities and metabolic character of traditional sour cream and butter. The objective of this study was to determine the bacterial microbiota and metabolic character of eight samples collected from herdsmen in Buryatia, Russia. Using single-molecule real-time (SMRT) sequencing techniques, we identified a total of 294 species and/or subspecies in 169 bacterial genera, belonging to 14 phyla. The dominant phylum was Firmicutes (81.47%) and the dominant genus was Lactococcus (59.28%). There were differences between the bacterial compositions of the sour cream and butter samples. The relative abundances of Lactococcus lactis, Lactococcus raffinolactis, and Acetobacter cibinongensis were significantly higher in sour cream than in butter, and the abundance of Streptococcus thermophilus was significantly lower in sour cream than in butter. Using a pure culture method, 48 strains were isolated and identified to represent seven genera and 15 species and/or subspecies. Among these isolates, Lactococccus lactis subsp. lactis (22.50%) was the dominant LAB species. Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry at elevated energy was used in combination with statistical methods to detect metabolite differences between traditional sour cream and butter. A total of 27,822 metabolites were detected in all samples, and Lys-Lys, isohexanal, palmitic acid, Leu-Val, and 2'-deoxycytidine were the most dominant metabolites found in all samples. In addition, 27 significantly different metabolites were detected between the sour cream and butter samples, including short peptides, organic acids, and amino acids. Based on correlation analyses between the most prevalent bacterial species and the main metabolites in sour cream, we conclude that there may be a connection between the dominant LAB species and these metabolites. This study combined omics techniques to analyze the bacterial diversity and metabolic character of traditional sour cream and butter, and we hope that our findings will enrich species resource libraries and provide valuable resources for further research on dairy product flavor.